CRISPR/Cas9 gRNA Vector
Design Guide

GenScript CRISPR gRNA/Cas9 Plasmids
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Putting CRISPR into Practice:With CRISPR/Cas9 genome editing, modified clonal cell lines can be derived within 2-3 weeks starting fromthe gRNA design stage, while transgenic animal strains can be created in a single generation. The following
workflows and case studies describe best practices on how to use CRISPR in your laboratory.

CRISPR/Cas9 Vector Design Workflow
CRISPR/Cas9-mediated gene editing can be performed in the following steps:
1. Determine Genetic Modification
Select the application for your experiment (Table 3).
Table 1:
GeneticApplication

Modification
Knock-out

Knock-in

Permanently remove genefunction
Generate a specific sequencechangeNuclease

gRNA

ActivityCas or Cas9n

Cas or Cas9n

Interference

Reduce gene expressiondCas-repressor

Activation

Increase gene expressiondCas-activator

gRNA targeting 5′ exon oressential protein domains
gRNA targeting region of interest

gRNA targeting gene promoter
elements
gRNA targeting gene promoter
elements

2. Select Expression System
CRISPR/Cas9 system components can be delivered in vivo using modified non-viral plasmid orviral vector or delivery systems (Table 2).
Table 2:
Expression SystemComponents

ApplicaƟonConstitutive or inducible Cas9
Plasmid Vector

Constitutive or inducible gRNA

Expression of Cas9 and gRNA

Reporter/selection marker

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Lentiviral Vector

Constitutive or inducible Cas9

Expression of Cas9 and gRNA

Constitutive or inducible gRNA

For infection ofReporter/selection marker

difficult-to-transfect cell types

Constitutive or inducible Cas9AAV Vector

Constitutive or inducible gRNA
Reporter/selection marker

Transient or stable expressionof SaCas9 and/or gRNA
For non-toxic infection ofdividing and non-dividing cells
Transient expression of CRISPR

Cas9 mRNA and gRNA

In vitro transcribed Cas9 mRNA and gRNA

sgRNA/Cas9 Ribonucleoprotein

Purified Cas9 protein and chemically synthesized

Transient expression of CRISPR(RNP) Complexes

single guide RNAgene editing components

gene editing components

Modified recombinant AAV particles are attractive for transduction because of their low immunogenicity
and capability to infect both quiescent and dividing cells. While AAV vectors are a preferred vehicle for invivo gene delivery, the size of the SpCas9 gene (>4 kb) exceeds the typical cargo limit of AAV vectors.
Solutions that have been developed to date include:• Creating transgenic animal lines that express Cas9, either constitutively or in an inducible manner, andthen to deliver only the guide RNAs and any necessary inducer at the time of the experiment.
• Developing a split-Cas9 system using split-inteins.
• Use smaller Cas9 orthologues from other species, such as Staphylococcus aureus (SaCas9), which
are small enough to be packaged along with a single guide RNA expression cassette into a single AAVvector.
Some of the most widely-used model systems for biomedical research are primary mammalian cell
cultures or hard-to-transfect cell lines in which transfection efficiency can be quite low. For these cell
types lentiviral vectors are preferred.
Guide RNAs may be also introduced via U6-gRNA cassette expression, but the cleavage efficiency is
typically lower than when gRNA is expressed from a plasmid. However, U6-gRNA cassettes may be usedfor rapid comparison of gRNA cleavage efficiencies so that the most optimal gRNA sequences can be
identified before subsequent cloning into your vector.
3. Gene Sequence Analysis
It is highly advisable to sequence the region of interest for the cell line or animal model you are using,rather than assuming it matches with the NCBI reference sequence for your species/strain. Allele number
may also vary depending on species/strain. Variations can result in reduced cleavage efficiency.
4. Select Sequence for Modification
Select specific genetic sequences for modification depending on your application (Table 1).

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For loss-of-function mutations, design gRNAs against early exons to optimize expression disruption andprevent the expression of truncated protein isoforms. Alternatively, targeting a functional site cangenerate a loss-of-function mutant. For genes with multiple splice variants, care should be taken toensure that a constitutive exon is targeted if the goal is to knock out all splice variants.
For applications using paired Cas9n, opposite strands of the genomic DNA should be targeted, with a
40-60 bp offset between PAM sequences.
For interference and activation applications, promoter elements within 200 bp of the transcription start siteshould be targeted.
5. Determine On/Off-Target Activity
Identify all PAM sequences within the region of interest: the PAM sequence will vary depending on theCas variant being used in the experiment (Table 3). A PAM sequence is required for targeting, so if none
are present, considering targeting a different location. The next 20 bps upstream of the PAM willcorrespond with your putative gRNA sequence. Be sure to check for off-target sites, locations within thegenome where partial homology is present, which can result in off-target cleavage.
Table 3:
Cas Variant

PAM Sequence

eSpCas9/SpCas9

NGG

SpCas9 VRER Variant

NGCG

SpCas9 EQR Variant

NGAG

SpCas9 VQR Variant

NGAN or NGNG

SaCas9NNGRRT

Cpf1

TTN

GenScript hosts free online human and mouse genome-wide databases developed by researchers at theBroad Institute. These databases can be searched and accessed here:
http://www.genscript.com/gRNA-database.html
GenScript also host a free online gRNA design tool developed by researchers at the Broad Institute. Thedesign tool can be accessed here:
http://www.genscript.com/gRNA-design-tool.html
6. Designing Knock-In Constructs
To introduce specific changes within the genome it is necessary to supply a donor template that can beused for HDR after Cas9 creates a DSB. HDR templates may be delivered as plasmids or as4

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single-stranded oligos (ssODN). To detect successful HDR and quantify knock-in efficiency, donor
templates are often designed to include several synonymous mutations that can be distinguished from thewild-type through sequencing. To prevent the cleavage of donor DNA after successful HDR, the donor
template should be designed with mutations in the PAM sequence.
As a general rule, eSpCas9/SpCas9 more efficient at mediating homologous recombination than Cas9nickase.
7. Synthesize gRNA/Cas9 Vectors
Once you have determined your expression system, Cas nuclease and gRNA sequence your customized
vectors can be cloned or commercially ordered.

CRISPR-mediated Gene Edi ng in Model Systems
CRISPR/Cas9-mediated genome editing has been successfully conducted in many different species andmodels (Table 6). Although the basic CRISPR/Cas9 components are the same regardless of the modelorganism, the delivery method varies widely, and choosing the most appropriate vector for your host is
critical for success.
Table 4:
HostPAM Sequence
Lipofection-based transfection of DNA plasmids

Mammalian Cells

Electroporation of DNA plasmids or RNP
Lenti or AAV virus-based transfection of DNA plasmids

Bacteria

Transformation of plasmids into competent cells

YeastElectroporation of plasmids and galactose induction of Cas9Mouse:
Germline Mutations

Direct injection into embryos
Electroporation into zygotesMouse:
Somatic Mutations

Direct injection of AAV into tissue of interest

Danio rerio

Direction injection into one-cell embryos

Drosophila melanogaster

Direct injection into embryo germline

Danio rerio

Direction injection into one-cell embryos

Caenorhabditis elegans

Direct injection into hermaphrodite germline

PlantsAgrobacterium-mediated transformation of gRNA/Cas9 vector

In vitro genome editing:For easy-to-transfect cell lines, plasmids encoding gRNAs and Cas9 can be delivered with high eﬃciency via lipofec on.
CRISPR plasmids typically contain selec on markers such as genes conferring an bio c resistance, or fluorescent proteins foreasy visualiza on via FACS.

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For diﬃcult-to-transfect cell lines or primary cells, len viral vectors are preferred. Guide RNAs may be delivered either via an
all-in-one plasmid that also encodes the Cas9 nuclease, or a separate plasmid that can be delivered into cells alreadyexpressing Cas9 (Figure 8).

In vivo genome editing:
CRISPR/Cas9 system components can be delivered to germ line cells to create heritable mutations.
Stable, homozygous mutations at multiple loci can be achieved in a single generation in mice.
CRISPR-mediated genome editing can similarly be used to generate precise mutations in somatic tissuesof adult animals, and to modify multiple genes at once in the same cells. These tools are especiallyvaluable for creating clinically relevant in vivo cancer models, because human tumors often contain acombination of gain-of-function mutations in oncogenes and loss-of-function mutations in tumor
suppressor genes42.

Verifica on of KO/KI and Oﬀ-target Eﬀects
To identify successful cases of CRISPR-mediated KO/KI and determine whether a loss-of-function or
gain-of-function mutation has occurred, mRNA and protein gene products should be analyzed.
Techniques such as quantitative PCR, Northern blotting, and Western blotting can be utilized to determine
whether mRNA and protein concentrations are depleted or molecular weights are changed.
For difficult-to-transfect cells, it can be sufficient just to show that high KO/KI efficiency has beenachieved, without isolating clones for confirmation. For these cases genome editing efficiency is typicallyassayed by next generation sequencing. A range of unique insertions and deletions will likely beobserved.
Best practices for managing off-target Cas9 activity:• Use at least two independent gRNA sequences in parallel to derive distinct clones. Models createdthrough genome editing with distinct gRNAs that share the on-target locus, but do not share off-target lociare an excellent way to create independent replicates.
• Isolate multiple, independent clonal cell populations for each gRNA used. The likelihood off-target DSBs
occurring at the same loci in independent clones is very low.
• Although few labs have the resources to do statistically powerful whole genome sequencing verification
protocols such as gUIDEseq, it is relatively easy to select the few predicted off-target sequences for eachgRNA you use and then sequence around those loci to ensure that off-target indels have not been
introduced.

To determine off-target effects, it is recommended to sequence predicted off-target sites, particularly thosewith matches in the “seed” region of the 20 mer recognition site, which lies adjacent to the PAM. Morerigorous reviews of off-target cleavage can be performed using whole-genome sequencing.

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